Sustainable chick cell line infected with marek&#39;s disease virus

ABSTRACT

A sustainable cell line of a Marek&#39;s disease herpesvirus (MDV) infected chicken cell line derived from chick embryo cells which are chicken helper factor (Chf) negative and which have been treated with N-methyl-N-nitro-N-nitrosoguansidine (MNNG) and then converted with the MDV which is able to infect avians in vivo. The cell line is useful for vaccine production and for determining the characteristics of the MDV under various conditions.

This is a divisional of copending application(s) Ser. No. 08/549,045filed on Oct. 27, 1995.

BACKGROUND OF THE INVENTION

1. Summary of the Invention

The present invention relates to a sustainable chicken cell lineinfected with Marek's disease virus (MDV). In particular, the presentinvention relates to a cell line which can be used as a virus vaccine,as a system in which to produce altered MDV strains, and which can beused to determine the affect of various reagents or growth conditions onthe cell line over time.

2. Description of Related Art

Marek's disease (MD) is a highly contagious lymphoproliferative diseaseof chickens, characterized by lymphocytic infiltration in visceralorgans, muscles, and peripheral nerves. The etiological agent of MD, anavian herpesvirus called Marek's disease virus (MDV), is highlyinfectious and cell associated (Calnek, B. W., and R. L. Witter, In"Diseases of Poultry: Marek's Disease" (B. W. Calnek, et al., Eds.), pp.342-385. Iowa State University press, Ames, Iowa (1991)). MDV replicatesin a productive restrictive manner in B-lymphocytes and cells growing intissue culture. Production of fully enveloped virus is restricted tofeather follicle epithelium of infected birds (Witter, R. L., et al., J.Natl. Cancer Inst. 49:1121-1130 (1972); Calnek, B. W., et al., AvianDis. 14:219-233 (1970)). MDV rapidly establishes a latent infection inT-lymphocytes, ultimately leading to malignant transformation andneoplastic disease (Shek, W. R., et al., J. Natl. Cancer Inst.70:485-491 (1983)). However, the precise relationship between latencyand transformation in MDV infected T-lymphocytes is unknown. Akiyama,Y., et al. (Continuous cell culture from lymphoma of Marek's disease.Biken J. 16:177-179 (1973)) first succeeded in establishing aT-lymphoblastoid cell line from MD-infected chickens. Since then, morethan 80 cell lines have been produced from MD lymphomas (Akiyama, Y., etal., Two cell lines from lymphomas of Marek's disease. Biken J.17:105-116 (1974); Powell, P. C., et al., Nature 251:79-80 (1974);Calnek, B. W., et al., Int. J. Cancer 21:100-107 (1978); Payne, L. N.,et al., Int. J. Cancer 28:757-766 (1981); Nazerian K., and R. L. Witter,J. Natl. Cancer Inst. 54:435-458 (1975)). Although suitable for somestudies, these cell lines are many passages removed from the originalevent(s) leading to their transformation.

Evidence suggests that viral genomes in MD-lymphoblastoid cell lines arepredominately integrated into cellular chromosomes, but episomal formsalso exist (Delecluse, H.-J., et al., J. Virol. 67:82-92 (1993)).Analysis of viral transcription in transformed lymphoblastoid cell lineshas revealed variable but limited transcriptional activity confined toapproximately 20% of the viral genome (Maray, T., et al., Virus Genes2:49-68 (1988)). MDV-specific transcripts in transformed lymphoblastoidcells are primarily derived from within long and short region terminalrepeats (TRL, and TRS respectively) and internal repeats (IRL and IRRrespectively). Little transcriptional activity is detected within eitherthe long unique (U_(L)) or short unique (U_(S)) regions (Sugaya, K., etal., J. Virol. 64:5773-5782 (1990)). MDV can be rescued from somelymphoblastoid cell lines by co-cultivation with primary or secondarychicken and duck embryo fibroblasts (CEF and DEF, respectively), whichsupport the lytic cycle of MDV in vitro (Schat, K. A., et al., Int. J.Cancer 44:101-109 (1989)). In addition, some lymphoblastoid cell lineswill induce MD upon injection into susceptible birds (Akiyama, Y., etal., Continuous cell culture from lymphoma of Marek's disease. Biken J.16:177-179 (1973); Nazerian, K., et al., Avian Diseases 21:69-76(1977)).

Since development of live virus Marek's disease vaccines in the late1970's, losses to Marek's disease have been significantly reduced(Calnek and Witter, In "Diseases of Poultry: Marek's Disease" (B. W.Calnek, H. J. Barnes, C. W. Beard, W. M. Reid, and H. W. Yoder, Jr.,Eds.), pp. 342-385. Iowa State University press, Ames IA (1991)). Themost widely used Marek's disease vaccines are live Turkey herpesvirus(HVT or serotype 3 MDV) or a bivalent mixture of HVT and the apathogenicserotype 2 strain of MDV (MDV-2). The bivalent mixture of HVT andserotype 2 MDV synergistically affords greater protection againstMarek's disease, especially in those situations where HVT is not fullyeffective (Witter, R. L., Protection by attenuated and polyvalentvaccines against highly virulent strain of Marek's disease virus. AvianPath. 11:49-62 (1982); Witter, R. L. and L. F. Lee, Polyvalent Marek'sdisease vaccines: safety, efficacy and protective synergism in chickenswith maternal antibodies. Avian Path. 13:75-92 (1984); Witter, R. L.,Principles of Vaccination. In "Marek's Disease: Scientific Basis andMethods of Control" (Payne, L. N., ed.) pp. 203-250. Marinus NijhoffPub., Boston, Mass. (1985)). Marek's disease vaccines are the mostwidely used vaccines in the poultry industry. Current Marek's diseasevaccines are either suspensions of infected chicken embryo fibroblasts(CEF) or cell-free virus suspensions made from sonicated CEF infectedwith vaccine strains of Marek's disease virus (MDV).

Two major difficulties in working with MDV are the strongly cellassociated nature of the virus and the lack a sustainable cell culturesystem amenable to productive (lytic) infections. Primary CEF and DEFare permissive for MDV replication. However, these cultures have afinite life span (approximately 3 weeks), thus necessitating passage ofinfected primary cells onto an uninfected cell monolayer to propagateMDV and to obtain sufficient quantities of virus with which to work.Such conditions also preclude establishment of one-step growthexperiments for effective temporal gene regulation studies. The finitelife span of CEF and DEF also make positive selection in mutagenesisstudies difficult.

The poultry industry has always recognized the need for continuous aviancell lines that could be used in producing Marek's disease vaccines andsimplify development of recombinant MDV vectors for polyvalent vaccines.Although many avian cell lines have been developed (Nazarian, K., Anupdated list of avian cell lines and transplantable tumors. Avian Path.12:527-544 (1987)), none of these can substitute for CEF cells invaccine production. Previous cell lines failed because they were eitherderived from virally transformed cells or, if derived from chemicallytransformed cells, the cells produced tumors when inoculated intochickens. Since there are no sustainable cell lines suitable forpropagating MDV, the MDV vaccine industry uses primary chicken embryofibroblasts (CEF) for production of vaccine virus (Churchill, A. E.,Production of Vaccines. In "Marek's Disease: Scientific Basis andMethods of Control" (Payne, L. N., ed.), pp. 251-265. Marinus NijhoffPublishing, Boston, Massachusetts (1985)). Since primary CEFs have afinite life span, they must be prepared every week, increasing costs forproducing MDV vaccines. For example, one major United States MDV vaccineproducer utilizes 25,000 chick embryos every week. It is estimated thatcosts associated with purchase and preparation of chick embryos accountsfor 40 to 45% of the total cost of MDV vaccine production. A significantreduction in MDV vaccine production costs could be realized if acontinuous cell line suitable for vaccine production were established.Requirements for such a cell line are: 1) The cell line can not bevirally transformed, 2) Chemically transformed cell lines must beincapable of inducing tumors in vaccinated chickens, and 3) Virus titersproduced by such a cell line must be equivalent (or nearly so) to titersobtained through infection of primary cells.

Development of a continuous cell line which would support MDVreplication would alleviate many of the difficulties associated with MDVexperimentation and vaccine production.

OBJECTS

It is therefore an object of the present invention to provide asustainable cell line for MDV. Further still, it is an object of thepresent invention to provide a method for producing the sustainable MDVcell line. Further still, it is an object of the present invention toprovide a method for infecting an avian to provide Marek's diseaseimmunity. Further still, it is an object of the present invention toprovide methods for producing the MDV cell line which is economical anda method for using the cell line which is effective. These and otherobjects will become increasingly apparent by reference to the followingdescription and the drawings.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A and 1B are photomicrographs showing uninfected and infectedcells. Monolayer of uninfected CHCC-OU2 cells display a cobblestoneappearance (FIG. 1A), similar to that seen with primary CEF and DEF. Atfour weeks post-infection (as set forth in Example 1) numerous plaquesconsistent with MDV infection were observed on monolayers of Md11p15/OU2cells (FIG. 1B).

FIG. 2 is an electrophoresis gel showing DNA from PCR amplification ofMDV-specific sequences. PCR amplification was carried out on DNAisolated from Md11 infected CHCC-OU2 cells (lanes 2-6). MDV-specificoligonucleotide primers (Table 1) were used to amplify particular MDVsequences, as set forth in Table 1.

Negative control (lane 1), ICP4 gene promoter sequences (lane 2), 900 bpregion of ICP4 coding sequence (lane 3), 1200 bp of BamHI L fragment(lane 4), gC gene promoter sequences (lane 5), US3 gene promotersequences (lane 6), and UL54 gene sequences (lane 7). Positions ofselected bands were obtained from a 1 kb ladder marker, which is notshown (Life Technologies, Inc., Gaithersburg, Md.).

FIG. 3 is an electrophoresis gel showing detection of viral DNA ininfected CHCC-OU2 cells. DNA was extracted from cells, digested withBamHI, electrophoresed on 0.8% agarose gels, transferred toHybond-N-Nylon membranes, hybridized to non-radioactive probe under highstringency conditions, and autoradiographed. Cloned MDV DNA BamHIfragments B, D, F, H, and I₂ were used as probes. Locations of eachfragment were determined by comparison to a DNA size standard (lambdaDNA digested with HindIII) and are indicated by an arrowhead to theleft.

FIGS. 4A to 4C are Western blots for detection of specific MDV proteins.Cell lysates from uninfected CEF (CEF), Md11p15 infected CEF (Md11p15),uninfected CHCC-OU2 cells (CHCC-OU2), and Md11p15 infected OU2 cells(MDV OU2.2) were resolved on 12.5% SDS-polyacrylamide gels andtransferred to nitrocellulose membranes, followed by immunodetectionusing specific antisera as described in Materials and Methods. In FIGS.4A and 4B, positions of molecular size markers (in Kilodaltons), areindicated. FIG. 4A shows detection of MDV-specific protein pp38 using amonoclonal antibody (generously provided by Dr. Lucy Lee, USDA-ADOL,East Lansing, Mich.). FIG. 4B shows detection of MDV-specific proteinpp14 using polyclonal antisera generated against pp14 fusion proteins(Hong, Y., and P. M. Coussens, J. Virol. 68:3593-3603 (1994)). FIG. 4Cprotein loading in each lane was verified by detection of α-actin usinga commercial antisera (Santa Cruz Biotechnology, Santa Cruz, Calif.).

FIG. 5 is an electrophoresis gel showing DNA from PCR amplification of850 bp pp38 gene segment. PCR amplification was carried out on DNAisolated from kidneys of birds injected with CHCC-OU2 (lanes 2 and 3),MDV OU2.2 (lanes 4-6), and Md11p16 infected CEF (lanes 7 and 8).Negative control (lane 1) included all reaction components excepttemplate DNA. Additional controls included DNA isolated from uninfectedCEF (lane 9), and DNA isolated from Md11p16 infected CEF as positivecontrol (lane 10). In each case (except negative control), 300 ng DNAwas used as template for PCR amplification using pp38 specific primers(Table 1). PCR products were analyzed on 12% agarose gels containing 10μg/ml ethidium bromide. Fragment sizes were determined relative to DNAsize standards, which are not shown (1 Kb Ladder, Life Technologies,Gaithersburg Md.).

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention relates to a sustainable Marek's disease virus(MDV) infected chicken cell line derived from chick embryo cells (CEC)which are chicken helper factor (Chf) negative and virus-free and whichhave been treated with N-methyl-N¹ -nitro-N-nitrosoguanidine (MNNG) andthen are infected with MDV which is able to infect avians in vivo.

Further, the present invention relates to a method for infecting anavian with Marek's disease virus (MDV) which comprises: providing avaccine produced by a sustainable Marek's disease virus (MDV) infectedchicken cell line derived from chick embryo cells (CEC) which arechicken helper factor (Chf) negative and virus-free and which have beentreated with N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and then areinfected with MDV which is able to infect avians in vivo; andinoculating the avian with the vaccine.

Finally, the present invention relates to an avian vaccine in dosageunit form containing a sustainable Marek's disease virus (MDV) infectedfibroblast cell line derived from chick embryo cells (CEC) which arechicken helper factor (Chf) negative and virus-free and which have beentreated with N-methyl-N¹ -nitro-N-nitrosoguanidine (MNNG) and then areinfected with MDV which is able to infect avians in vivo.

The new MDV infected cell lines are "sustainable" which means that itcan be maintained in culture for long periods of time (at least nineteenmonths) without requiring passage. This is a great improvement overmultiple passaging in CEC cells as in the prior art.

The virus free cell line CHCC-OU2 is maintained on deposit at MichiganState University and by the USDA and is a known cell line and is freelyavailable upon request. The CHCC-OU2 cell line was deposited under theterms of the Budapest Treaty at the American Type Culture Collection(ATCC), 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A. on Feb. 26,1997 under the accession number CRL-12302. The CHCC-OU2 cell line willbe irrevocably available from the ATCC for the life of the patent.Similarly MDV infected CHCC-OU2 cells deposited under the BudapestTreaty on Sep. 28, 1995, as ATCC CRL-11985 (MDVOU2.2) and is availableupon request by name and number. The MDV infected CHCC-OU2 cells will beirrevocably available from the ATCC for the life of the patent.

The MDV infected cell line can be used to produce a vaccine so long asthe virus is non-oncogenic. The MDV infected cell line can also be usedto test various agents or conditions which suppress or enhance cellresponse, such as repressing tumor production.

The preferred MDV is a virus selected from Serotypes 1, 2 or 3. Serotype3 MDV is also referred to as turkey herpesvirus (HVT). The MDV can be arecombinant or deletion mutant which is used as a vaccine or for otheruses.

The present invention particularly relates to continuous chickfibroblast cell lines (MDV OU 2.1 and MDV OU2.2), stably infected withMDV strain Md11 at passage level 15. MDV OU2.1 and MDV OU2.2 cells growcontinuously in culture and, once confluent, display plaquescharacteristic of MDV infection. MDV OU2.1 and MDV OU2.2 cells can beused to transfer infection to CEF, and produce classic symptoms of MD insusceptible birds. MDV OU2.2 cells have remained viable and continue toproduce MDV after cryogenic storage and continuous culture for over 19months.

As shown in the following Example 1, Southern blot and PCR analysesdemonstrate that these cell lines harbor MDV DNA. Western blot analysesindicate that MDV OU2.2 cells express at least a limited set of viralproteins, pp 38 and pp14, similar to that seen in MDV lymphoblastoidcells. Presence of distinct plaques in confluent MDV OU2.2 cellmonolayers is consistent with cytolytic semi-productive infection,similar to that observed in primary CEF. MDV OU2.2 cells are capable oftransferring MDV infection to primary CEF cultures and inducing clinicalsigns of Marek's disease (MD) in susceptible birds. MDV OU2.2 cells havemaintained a MDV positive phenotype for over 19 months of activeculture.

EXAMPLE 1 MATERIALS AND METHODS

Cells and Virus

Preparation, propagation, and infection of CEF cells with MDV wereperformed as described previously (Glaubiger, C., et al., J. Virol.45:1228-1234 (1983); Coussens, P. M., and L. F. Velicer, J. Virol.62:2373-2379 (1988)). The very virulent MDV strain Md11 was used in thisstudy at cell culture passage level 15 (Md11p15). CHCC-OU2 cells (Ogura,H., et al., Acta Med. Okayama 41:141-143 (1987)) were obtained from Dr.Donald Salter, Avian Disease and Oncology Laboratories (ADOL), U.S.department of Agriculture (USDA), East Lansing, Mich., and were culturedin Leibovitz L15-McCoy 5A (LM) (Gibco, Inc., Grand Island, N.Y.) media(carbon, nitrogen source) supplemented with 10% fetal bovine serum (FBS)and 2% tryptose phosphate broth (TPB) at 37° C. in a humidifiedatmosphere containing 5% Co₂.

CHCC-OU2 cells were infected with MDV strain Md11p15 by combining5.0×10⁷ CHCC-OU2 cells with 2.0×10⁷ Md11p15 infected CEF prior toplating on 150 mm culture dishes in LM medium supplemented with 4% calfserum (CS). Co-cultivation of CHCC-OU2 cells with Md11 infected CEFcells was continued for four passages. Cells from each of these passageshave been preserved at -135° C. in freezing media (LM media (LifeTechnologies, Gaithersburg, MD, McCoy's 5A medium) supplemented with 20%CS and 10% dimethyl sulfoxide (DMSO). At four passages post-infection,numerous plaques (approximately 100 plaques per 150 mm culture dish),characteristic of MDV infections in CEF cells, were observed. Two ofthese plaques were isolated using sterile cloning cylinders. Cylinderswere placed on top of individual plaques, cells were trypsinized andaspirated from the cloning cylinders. Aspirated cells were transferredto 35 mm culture dishes containing LM media supplemented with 4% CS forexpansion. During expansion, cells were not allowed to become confluentand media was changed every 48 to 72 hours. Expanded clones weredesignated MDV OU2.1 and MDV OU2.2.

Preparation of cellular DNA, Southern blot analysis, and PCR

Total cellular DNA was extracted from uninfected and MDV-infectedCHCC-OU2 cells by standard methods (Sambrook, J., et al., Molecularcloning a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1989)). Restriction enzymes (Boehringer MannheimBiochemicals, Indianapolis, Ind.) were used according to themanufacturers recommendation. DNA was digested, electrophoresed through0.8% agarose gels and transferred to Hybond-N nylon membranes (AmershamCorp., Arlington Heights, Ill.) by Southern blotting (Southern, E. M.,Detection of specific sequences among DNA fragments separated by gelelectrophoresis. J. Mol. Biol. 98:503 (1975)). Probes werenon-radioactively labeled (Digoxigenin-11-dUTP) using a random primerlabeling kit (Boehringer Mannheim Biochemicals, Indianapolis, Ind.).

Total cellular DNA was also used as template for PCR amplification ofMDV specific sequences. Primers used and expected fragment sizes areindicated in Table 1.

                                      TABLE 1    __________________________________________________________________________    Sequence of MDV-specific oligonucleotide primers used in PCR    amplification.                                         Expected    Primer Sequences*              Locus Size (kbp)    __________________________________________________________________________    GTAGTGAAATCTATACCTGGG (SEQ ID NO. 1)                                   gC gene                                         0.3    GTGTCTAGAGAGGGAAGATATGTAGAGGGTTAC (SEQ ID NO. 2)                                   promoter    ATGGAATTCGAAGCAGAACAC (SEQ ID NO. 3)                                   pp38 gene                                         0.85    CTCCAGATTCCACCTCCCCAGA (SEQ ID NO. 4)    TGCTAATTGTGGCTCC (SEQ ID NO. 5)                                   ICP4 gene                                         0.9    GGTGCTTCCATCTCGGC (SEQ ID NO. 6)    GATCTAGACGTTTCTGCCTCCGGAGTC (SEQ ID NO. 7)                                   US3 gene                                         0.6    GCAAGCTTCAACATCTTCAAATAGCCGCAC (SEQ ID NO. 8)                                   promoter    GTCTAGACGCGATAGCGAGTTGTTGGACCC (SEQ ID NO. 9)                                   ICP4 gene                                         1.1    GGAAGCTTTATTAAGGGAGATTCTACCC (SEQ ID NO. 10)                                   promoter    GTGAAAGAGTGAACGGGAAG (SEQ ID NO. 11)                                   BamHI L                                         1.2    CGTCAAAGCGATAATAGGC (SEQ ID NO. 12)                                   fragment    CCGGGGATCCCGAAATGTCGTTAGAACATC (SEQ ID NO. 13)                                   UL54 gene                                         1.1    CGGGGTCGACTAAGGCAAATAGGCACGC (SEQ ID NO. 14)    __________________________________________________________________________     *Primer sequences are written as 5' to 3', left to right. In each case,     the upper primer represents the upstream sequence while the lower primer     represents the downstream sequence. They are set forth in Appendices 1 to     14.

Briefly, 300 ng of total cellular DNA was combined with 25 mM each dNTP(dATP, dCTP, dGTP, and dTTP), 20 mM of each appropriate primer pair, 10μl of 10X PCR reaction buffer (Perkin Elmer Cetus, Norwalk, Conn.), and2.5 Units Taq polymerase (Perkin Elmer Cetus, Norwalk, Conn.). PCRreactions were performed using a GeneAmp 9600 thermal cycler (PerkinElmer Cetus, Norwalk, Conn.) as follows: 35 cycles of 95° C. for 20 sec,56° C. for 20 sec. and 72° C. for 30 sec. Two controls, one without DNAand one with uninfected CHCC-OU2 DNA were included in each experiment.High molecular weight DNA isolated from uninfected CEF and CEF infectedwith Md11p16 were used as controls for specific amplification. PCRproducts were purified using the Wizard PCR prep kit (Promega Inc.,Madison, Wis.) as recommended by the manufacturer) and analyzed on 12%agarose gels.

Western immunoblot analysis

Cultured cells were collected and sonicated using a Sonifier celldisrupter model 350 (Branson Ultrasonic Corporation, Danbury, Conn.).Proteins (20 μg) from each cell type were separated on 12.5%polyacrylamide/1% SDS gels. Separated proteins were electrophoreticallytransferred to nitrocellulose membranes. Membranes were blocked with 5%nonfat milk and probed with antibodies to MDV proteins: pp14 (Hong, Y.,and P. M. Coussens, J. Virol. 68:3593-3603 (1994)), and pp38 (Cui, Z.,et al., J. Virol. 65:6509-6515 (1991)). Immune complexes were detectedby incubation with a donkey anti-mouse or anti-rabbit immunoglobulinconjugated with horseradish peroxidase. Detection was performed using anECL Western blot kit (Amersham Corp., Arlington Heights, Ill.) accordingto the manufacturer's recommendations and exposed to X-ray film. Proteinsizes were estimated by comparison to prestained protein molecularweight standards (Bio-Rad, Richmond, Calif.) electrophoresed on the samegel. Inoculation of chickens with cells and virus.

In vivo experiments were performed using specific pathogen free chickens(line 15I₅ ×7₁) obtained from the Avian Disease and Oncology Laboratory,U.S. Department of Agriculture, East Lansing, Mich. Three groups ofchicks each at one day of age, were inoculated intraperitoneally with(1) uninfected CHCC-OU2 cells, (2) 1000 plaque forming units (PFU) ofMd11p16 in CEF, and (3) 1000 PFU of MDV infected OU2 cells (MDV OU2.2).The first and second groups served as controls (negative and positive,respectively).

Birds were euthanized and necropsied upon signs of morbidity. Blood wascollected for isolation of peripheral blood lymphocytes andco-cultivation with CEF as an assay for viable virus. Kidney, spleen,and liver were harvested for DNA isolation and histological evaluation.Total tissue-specific DNA was isolated and used as template for PCRamplification of MDV sequences employing primer sets detailed in Table1.

RESULTS

Infection of CHCC-OU2 cells with Md11p15:

Although CHCC-OU2 cells are chemically immortalized, they are notmalignantly transformed, maintain contact inhibition, and exhibit manymorphological features of normal chick fibroblasts. CHCC-OU2 cells wereco-cultivated with Md11p15 infected CEF cells. Cytopathic effect (CPE),characterized by formation of spherical cells loosely attached to thesubstratum, was first observed on co-cultivation cell monolayers at twoweeks post-infection. The majority of these regions were characterizedas "microplaques", consisting of relatively small clusters of roundedcells. CPE was slow in developing and expanding. Fully developed plaquesconsisting of syncytia and extended regions of rounded, looselyattached, cells were not visible until four weeks post-infection (FIG.1). The appearance of visible plaques 14 days post-infection and thedependence of plaque formation on the cells reaching confluency andcontact inhibition was not expected. By comparison, a typical CEFmonolayer infected with MDV strain Md11 will develop readily visibleplaques in 5-7 days post-infection with complete destruction of themonolayer within 10-14 days. Plaque formation in CEF is independent ofthe confluency of the cell monolayer. After four weeks ofco-cultivation, cells were cryogenically preserved at -135° C. for twoweeks. Cell cultures were re-established from frozen cells by combininginfected (Md11p15/OU2) and uninfected CHCC-OU2 cells. Plaques consistentwith MDV infection were not observed until cells reached confluency,approximately 14 days post-plating.

Detection of MDV DNA in infected CHCC-OU2 cells

Polymerase chain reaction (PCR) was used as an initial assay forpresence of MDV DNA in infected CHCC-OU2 cells. Three hundred ng oftotal DNA from Md11p15/OU2 and uninfected CHCC-OU2 cells was used astemplate for PCR amplification with several primer pairs correspondingto various MDV genes, as described in Materials and Methods. Bands ofappropriate sizes (Table 1) were obtained in reactions with Md11p15/OU2DNA but not from uninfected OU2 DNA templates (FIG. 2). Reactionscontaining DNA isolated from uninfected CEF and Md11 infected CEF wereused as negative and positive controls, respectively (data not shown).

Results of PCR analyses indicated that MDV DNA was present in infectedCHCC-OU2 cultures. Although unlikely, given our extended cultureconditions, PCR analysis could have detected MDV DNA from residualMd11p15 infected CEF cells. In addition, PCR analyses do not providecritical information on integrity of MDV DNA in Md11p15/OU2 cells. Toaddress these concerns, MDV DNA in infected CHCC-OU2 cells was analyzedby Southern blot hybridization using a cocktail of MDV BamHI fragments(B, F, H, and I₂) as a probe. Total genomic DNA isolated from Md11p16infected CEF and Md11p15/OU2 cells contained MDV specific fragmentscorresponding to BamHI fragments B, F, H and I₂. As expected, similarfragments were not detected in DNA isolated from uninfected CEF orCHCC-OU2 cells (FIG. 3).

Establishment of infected cell lines

Although culture conditions and freeze-thaw cycles should haveeliminated most of the original CEF cells used for establishinginfection, it was possible that residual CEF cells were contributing toMDV specific DNA detected in our Md11p15/OU2 cultures. To address thisconcern, isolation and expansion of individual plaques from infectedCHCC-OU2 cultures was initiated. Two Md11p15/OU2 cell lines (MDV OU2.1and MDV OU2.2) were established by plaque isolation and expansion asdescribed in Materials and Methods. Despite arising from distinctplaques, both cell lines exhibited initial growth characteristicsindistinguishable from uninfected CHCC-OU2 cells. Plaques characteristicof MDV infection were only observed in MDV OU2.1 and MDV OU2.2 cellcultures after confluency had been reached at 10 to 14 dayspost-plating.

To confirm infectious virus could be rescued from MDV OU2.1 and MDVOU2.2 cultures, cells from each isolate were used as inoculum to infectCEF cells by combining 1×10⁶ MDV OU2.1 or MDV OU2.2 with 5×10⁷ secondaryCEF. Plaque consistent with MDV infection of CEF cells were visiblewithin 5 days post co-cultivation. In contrast, no plaques were evidentin control plates containing 1×10⁶ uninfected CHCC-OU2 cells and 5×10⁷CEF cells.

Detection of viral proteins expressed in MDV OU2.2 cells.

To verify that MDV OU2.2 cells indeed supported replication and growthof MDV, detection of MDV proteins was initiated. Monoclonal antibodiesto pp38 (generous gift from Dr. Lucy Lee, USDA-ADOL, East Lansing,Mich.), detected a band of approximately 38 kDa in extracts fromconfluent MDV OU2.2 cells and Md11p16 infected CEF, but not in extractsfrom uninfected CHCC-OU2 or CEF cells (FIG. 4A). Polyclonal antisera topp14, an MDV-specific immediate-early phosphoprotein (Hong, Y., et al.,J. Virol. 68:3593-3603 (1994)) also reacted with an appropriately sizedpolypeptide in extracts from confluent MDV OU2.2 cells and Md11p16infected CEF cells but not in uninfected cell extracts (FIG. 4B). Apolyclonal antisera to α-actin was used to ensure similar amounts ofprotein were analyzed in each lane (FIG. 4C). Taken together, results ofPCR amplification, Southern hybridization, and Western blot analysisindicated that MDV OU2.2 cells represent a continuous anchoragedependent cell line which harbors MDV and is permissive forsemi-productive infection.

MDV OU2.2 cells induce MD in susceptible chickens

Marek's disease can be experimentally induced by injection of MDVinfected cells into susceptible birds. MDV lymphoblastoid cell linessuch as MSB-1 are also able to induce MD in susceptible birds followingintraperitoneal injection. Also, most commercially available MDVvaccines are comprised of CEF cells infected with vaccine strains ofMDV. The infectious but apathogenic vaccine strains of MDV aretransferred to birds after inoculation where it replicates, inducinghumoral and cell mediated responses. To determine if MDV OU2.2 cellscould transfer MDV to birds in a similar manner, line 15I₅ ×7₁ chickenswere inoculated with uninfected CHCC-OU2 cells, MDV OU2.2 cells, orMd11p16 infected CEF cells at one day of age. Chickens injected witheither Md11p16 infected CEF or with MDV OU2.2 cells developed classicalsigns of MD (reduced growth and paralysis of neck, wings, and legs) asearly as 10 days post-infection. In contrast, negative control chickensinjected with uninfected CHCC-OU2 cells showed no clinical signs of MD,even at 12 weeks of age.

To confirm presence of MDV in infected birds, peripheral bloodlymphocytes (PBLs) were isolated from blood collected at various timespost-inoculation and seeded onto secondary CEF. Plaques consistent withMDV infection were observed on CEF monolayers at 4 days post-culture onplates seeded with PBLs isolated from birds injected with Md11p16infected CEF or MDV OU2.2 cells. No plaques were observed on plates ofCEF cells mixed with PBLs obtained from control birds injected withuninfected CHCC-OU2 cells.

To further confirm replication of MDV in infected birds, total cellularDNA isolated from infected bird kidneys was used as template for PCRamplification using primers specific for the MDV pp38 gene. Consistentwith the presence of MDV DNA an 850 bp fragment was amplified using DNAisolated from kidneys of birds injected with Md11p16 infected CEF or MDVOU2.2 cells. In contrast, similar bands were not detected when DNA frombirds injected with CHCC-OU2 cells was used as template (FIG. 5).

Histological evaluation revealed lymphocyte infiltration and early,active lymphomas in various tissues from birds injected with CEF/Md11p16and MDV OU2.2, whereas CHCC-OU2 inoculated birds showed no signs of MDat the microscopic level. Taken together, these results clearlydemonstrate that MDV OU2.2 cells contain MDV and that the virus may betransferred to birds via intraperitoneal injection.

One of the major difficulties in working with MDV is lack of asustainable cell culture system for virus growth and selection. Althoughprimary CEF and DEF are permissive for MDV replication, primary culturesare characterized by slow growth and limited life span. These factorsnecessitate continual passage of infected cells onto uninfected cells inorder to obtain sufficient quantities of virus with which to work. Inaddition, CEF and DEF must be prepared on a regular basis from 10 or 11day old chick embryos, adding significantly to the expense anddifficulty of culturing MDV.

The CHCC-OU2 cell line is an immortalized fibroblastic cell line derivedfrom chick embryo cells (Ogura, H., et al., Acta Med. Okayama 41:141-143(1987)). CHCC-OU2 are not oncogenic, based on the fact that CHCC-OU2cells failed to produce tumors when injected into syngeneic chickens(Ogura, H., et al., Acta Med. Okayama 41:141-143 (1987)). In additionCHCC-OU2 are virus free and susceptible to avian retrovirus infection(avian sarcoma viruses of subgroups A, B, and C). Newcastle diseasevirus also replicates well in CHCC-OU2 cell cultures (Ogura, H, et al.,Acta Med. Okayama 41:141-143 (1987)).

As can be seen from Example 1, the CHCC-OU2 cells are suitable as asustainable cell culture system for replication and study of MDV. Theinitial phase of CHCC-OU2 infection with MDV was slow and characterizedby a low number of visible plaques. Fully developed plaques were firstobserved at four weeks post-infection. At this time, plaques wereclearly visible and quite abundant (approximately 100 plaques/150 mmtissue culture plate). In subsequent passages, plaques were visibleevery 10-14 days in culture.

Southern blot and PCR analyses confirmed that semi-clonal cell lines,MDV OU2.1 and MDV OU2.2, indeed harbor MDV DNA. Fragments detected bySouthern blot hybridization were similar in size to those detected inMd11p15 infected CEF, indicating that no gross structural rearrangementshad occurred. In addition, fragments detected in MDV OU2.2 cell DNArepresent diverse regions of the MDV genome, including the unique long(BamHI B and F), terminal repeat long (BamHI D), and internal repeatlong (BamHI H and I₂) segments. Intensity of these fragments suggeststhat MDV OU2.2 cells allow MDV DNA replication, as it is highly unlikelythe observed amount of DNA would arise from residual CEF cells used toestablish initial infections.

Western blot analyses demonstrated that MDV OU2.2 cells express at leasta limited set of viral proteins, pp38 and pp14. Despite numerousattempts, we were unable to detect MDV glycoproteins gC, gB, gE, and gIby western blot analyses. Each of the particular antisera employed wasable to detect the respective protein in Md11p15 infected CEF cellextracts. Thus, results of western blot analyses are consistent with MDVexisting in MDV OU2.2 cells as a latent infection, similar to that seenin MDV lymphoblastoid cells. However, presence of distinct plaques inMDV OU2.2 cell monolayers is not consistent with latent infection asthis would imply cytolytic activity related to MDV infection. However,presence of distinct plaques in confluent MDV OU2.2 cell monolayersimply cytolytic activity related to MDV infection is dependent oncontact inhibition.

The in vivo and in vitro experiments demonstrated that in all respectsMDV propagated in OU2 cells was structurally and biologicallyindistinguishable from MDV propagated in CEF. Preliminary experimentswith vaccine viruses, SB1 and FC126, indicate that these virusesreplicate in the same manner as Md11p15 in OU2 cells. These experimentsalso indicated that these infected OU2 cells, like OU2 cells infectedwith Md11p15, are infectious and can transfer virus to CEF. Thepreliminary data with the vaccine strains of MDV and the in vivo data ofMd11p15 suggest that vaccines comprised of OU2 cells infected withvaccine strains of MDV will be just as effective as the current vaccinescomprised of CEF infected with vaccine strains of MDV.

EXAMPLE 2

CHCC-OU2 cells were infected with MDV serotype-2 vaccine strain SB1 byinfecting CHCC-OU2 monolayers comprised of 4×10⁶ cells on 100 mm culturedishes with 3.1×10⁶ SB1 infected CEF. Co-cultivation was continued forfour passages. At every passage, progressively more plaques,characteristic of MDV infections, were observed.

EXAMPLE 3

CHCC-OU2 cells were infected with MDV serotype-3 (HVT) vaccine strainFc126 by infecting CHCC-OU2 monolayers comprised of 4×10⁶ cells on 100mm culture dishes with 3.3×10⁶ HVT infected CEF. Co-cultivation wascontinued for four passages. At passage three, plaques, characteristicof MDV infections, were observed.

In vivo experiments clearly demonstrate that MDV OU2.2 cells are capableof transferring MDV infection to CEF monolayer cultures and inducingclinical signs of MD in susceptible birds. Birds injected with eitherMDV OU2.2 or Md11p16 infected CEF developed clinical signs of MD,characterized by a marked decrease in growth rate and paralysis of legs,wings, and neck. PCR analysis of tissues, including kidney and spleen,demonstrated that MDV was present in remote tissues of birds injectedwith MDV OU2.2 cells. In addition, PBLs isolated from birds injectedwith MDV OU2.2 cells were able to transfer infection to CEF monolayers.In contrast, no evidence of tumor formation or viremia was observed inbirds inoculated with uninfected CHCC-OU2 cells.

The in vivo experiments clearly demonstrate that MDV infected CHCC-OU2cells can be used to establish infections in susceptible birds, aquality of considerable importance for MDV vaccine development andproduction of MDV mutants by positive selection.

The foregoing description is only illustrative of the present inventionand the present invention is limited only by the hereinafter appendedclaims.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 14    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 Base Pairs    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (ii) MOLECULE TYPE:    (A) DESCRIPTION: Synthetic DNA    (iii) HYPOTHETICAL: No    (iv) ANTI-SENSE: No    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Marek's Disease Virus    (vii) IMMEDIATE SOURCE:    (A) LIBRARY:    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:    GTAGTGAAATCTATACCTGGG21    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 33 Base Pairs    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (ii) MOLECULE TYPE:    (A) DESCRIPTION: Synthetic DNA    (iii) HYPOTHETICAL: No    (iv) ANTI-SENSE: No    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Marek's Disease Virus    (vii) IMMEDIATE SOURCE:    (A) LIBRARY:    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:    GTGTCTAGAGAGGGAAGATATGTAGAGGGTTAC33    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 Base Pairs    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (ii) MOLECULE TYPE:    (A) DESCRIPTION: Synthetic DNA    (iii) HYPOTHETICAL: No    (iv) ANTI-SENSE: No    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Marek's Disease Virus    (vii) IMMEDIATE SOURCE:    (A) LIBRARY:    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:    ATGGAATTCGAAGCAGAACAC21    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 22 Base Pairs    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (ii) MOLECULE TYPE:    (A) DESCRIPTION: Synthetic DNA    (iii) HYPOTHETICAL: No    (iv) ANTI-SENSE: No    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Marek's Disease Virus    (vii) IMMEDIATE SOURCE:    (A) LIBRARY:    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:    CTCCAGATTCCACCTCCCCAGA22    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 16 Base Pairs    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (ii) MOLECULE TYPE:    (A) DESCRIPTION: Synthetic DNA    (iii) HYPOTHETICAL: No    (iv) ANTI-SENSE: No    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Marek's Disease Virus    (vii) IMMEDIATE SOURCE:    (A) LIBRARY:    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:    TGCTAATTGTGGCTCC16    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 Base Pairs    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (ii) MOLECULE TYPE:    (A) DESCRIPTION: Synthetic DNA    (iii) HYPOTHETICAL: No    (iv) ANTI-SENSE: No    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Marek's Disease Virus    (vii) IMMEDIATE SOURCE:    (A) LIBRARY:    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:    GGTGCTTCCATCTCGGC17    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE 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Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (ii) MOLECULE TYPE:    (A) DESCRIPTION: Synthetic DNA    (iii) HYPOTHETICAL: No    (iv) ANTI-SENSE: No    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Marek's Disease Virus    (vii) IMMEDIATE SOURCE:    (A) LIBRARY:    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:    GTCTAGACGCGATAGCGAGTTGTTGGACC29    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 28 Base Pairs    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (ii) MOLECULE TYPE:    (A) DESCRIPTION: Synthetic DNA    (iii) HYPOTHETICAL: No    (iv) ANTI-SENSE: No    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Marek's Disease Virus    (vii) IMMEDIATE SOURCE:    (A) LIBRARY:    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:    GGAAGCTTTATTAAGGGAGATTCTACCC28    (2) INFORMATION FOR SEQ ID NO:11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 Base Pairs    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (ii) MOLECULE TYPE:    (A) DESCRIPTION: Synthetic DNA    (iii) HYPOTHETICAL: No    (iv) ANTI-SENSE: No    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Marek's Disease Virus    (vii) IMMEDIATE SOURCE:    (A) LIBRARY:    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:    GTGAAAGAGTGAACGGGAAG20    (2) INFORMATION FOR SEQ ID NO:12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 Base Paris    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (ii) MOLECULE TYPE:    (A) DESCRIPTION: Synthetic DNA    (iii) HYPOTHETICAL: No    (iv) ANTI-SENSE: No    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Marek's Disease Virus    (vii) IMMEDIATE SOURCE:    (A) LIBRARY:    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:    CGTCAAAGCGATAATAGGC19    (2) INFORMATION FOR SEQ ID NO:13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 Base Pairs    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (ii) MOLECULE TYPE:    (A) DESCRIPTION: Synthetic DNA    (iii) HYPOTHETICAL: No    (iv) ANTI-SENSE: No    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Marek's Disease Virus    (vii) IMMEDIATE SOURCE:    (A) LIBRARY:    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:    CCGGGGATCCCGAAATGTCGTTAGAACATC30    (2) INFORMATION FOR SEQ ID NO:14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 28 Base Pairs    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (ii) MOLECULE TYPE:    (A) DESCRIPTION: Synthetic DNA    (iii) HYPOTHETICAL: No    (iv) ANTI-SENSE: No    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Marek's Disease Virus    (vii) IMMEDIATE SOURCE:    (A) LIBRARY:    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:    CGGGGTCGACTAAGGCAAATAGGCACGC28    __________________________________________________________________________

We claim:
 1. A method for infecting an avian with Marek's disease virus(MDV) which comprises:(a) providing a vaccine produced by an immortalMDV infected chicken cell line maintained as a monolayer whichreplicates continuously in cell culture, is contact-inhibited, andproduces infectious MDV when cell culture is confluent, and is able totransfer MDV to avians in vivo which consists of chick embryo cells(CEC) which are chicken helper factor (Chf) negative and virus-free andwhich have been treated with N-methyl-N-nitro-N-nitrosoguanidine (MNNG)to render an immortalized cell line that is not malignantly transformedand is contact-inhibited which is then infected with MDV selected fromthe group consisting of serotype 1, serotype 2, or serotype 3 MDVwherein serotype 3 is a turkey herpesvirus (HVT) to establish thesustainable MDV infected chicken cell line; and (b) inoculating theavian with the vaccine.
 2. The method of claim 1 wherein the inoculationis by injection.
 3. The method of claim 1 wherein the MDV is a virusused for the preparation of a virus vaccine to produce immunity.
 4. Themethod of claim 1 wherein the MDV is a virus selected from the groupconsisting of serotype 1, serotype 2, or serotype 3 MDV wherein serotype3 is a turkey herpesvirus (HVT).
 5. The method of claim 1 wherein theMDV is a genetically engineered MDV comprising one or more foreign genesinserted into a site in MDV.
 6. The method of claim 1 wherein the MDVcomprises one or more foreign genes which have been deleted.
 7. Themethod of claim 1 wherein a continuously replicating Marek's diseaseherpesvirus cell line deposited as ATCC CRL-11985 containing Marek'sdisease strain Md11.
 8. The method of claim 1 wherein the CECfibroblasts is CHCC-OU2.
 9. The method of claim 1 wherein the vaccinecontains the sustainable MDV cell line.
 10. An avian vaccine in dosageunit form containing an immortal Marek's disease virus (MDV) infectedfibroblast cell line which replicates continuously in cell culture, iscontact-inhibited, and produces infectious MDV when cell culture isconfluent, and is able to transfer MDV to avians in vivo which consistsof chick embryo cells (CEC) which are chicken helper factor (Chf)negative and virus-free and which have been treated with N-methyl-N¹-nitro-N-nitrosoguanidine (MNNG) to render an immortalized cell linethat is not malignantly transformed and is contact-inhibited which isthen infected with MDV selected from the group consisting of serotype 1,serotype 2, or serotype 3 MDV wherein serotype 3 is a turkey herpesvirus(HVT) to establish the sustainable MDV infected chicken cell line. 11.The vaccine of claim 10 wherein the MDV is a virus used for thepreparation of a virus vaccine to produce immunity.
 12. The vaccine ofclaim 10 wherein the MDV is a virus selected from the group consistingof serotype 1, serotype 2, or serotype 3 MDV wherein serotype 3 is aturkey herpesvirus (HVT).
 13. The vaccine of claim 10 wherein the MDV isa genetically engineered MDV comprising one or more foreign genesinserted into a site in MDV.
 14. The vaccine of claim 10 wherein the MDVinfected cell line comprises a recombinant MDV having one or more genesdeleted.
 15. The vaccine of claim 10 wherein a continuously replicatingMarek's disease herpesvirus cell line deposited as ATCC CRL-11985Marek's disease strain Md11.
 16. The vaccine of claim 10 wherein the CECfibroblasts is CHCC-OU2.